Is-targeting system for gene insertion and genetic engineering in deinococcus bacteria

ABSTRACT

The present invention relates to methods and compositions for chromosome integration of nucleic acids into Deinococcus bacteria. The invention more particularly relates to IS-mediated multicopy gene insertion or chromosome engineering in Deinococcus bacteria, the resulting bacteria, and the uses thereof.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a divisional of U.S. application Ser. No.15/039,867, filed May 27, 2016, which is the U.S. national stageapplication of International Patent Application No. PCT/EP2014/078858,filed Dec. 19, 2014.

The Sequence Listing for this application is labeled “Seq-List.txt”which was created on Jan. 23, 2015 and is 18 KB. The entire content ofthe sequence listing is incorporated herein by reference in itsentirety.

The present invention relates to methods and compositions for chromosomeintegration of nucleic acids into Deinococcus bacteria. The inventionmore particularly relates to IS-mediated multicopy gene insertion or IStargeting methods for chromosome engineering in Deinococcus bacteria,the resulting bacteria, and the uses thereof.

INTRODUCTION

Deinococcus is a gram positive bacterium that was isolated in 1956 byAnderson and collaborators. This extremophile organism is resistant toDNA damage by UV and ionizing radiations or by cross-linking agent(mitomycin C) and is tolerant of desiccation. WO01/023526 shows theunusual resistance of Deinococcus to radiation and further proposestheir engineering and use in bioremediation. WO2009/063079 shows thatDeinococcus bacteria can resist solvents and transform biomass togenerate biofuels. WO2010/130806 further discloses recombinantDeinococcus strains wherein ethanol biosynthesis genes have beeninserted. These recombinant strains exhibit improved performance in theproduction of ethanol.

The present invention discloses novel compositions and methods forgenetically modifying Deinococcus bacteria. More specifically, theinvention provides improved Insertion Sequence-based methods forgenetically modifying Deinococcus bacteria.

SUMMARY OF THE INVENTION

The invention relates to methods and constructs for gene recombinationor chromosome engineering in Deinococcus bacteria. More specifically,the invention relates to IS-based methods and constructs for geneinsertion or amplification in Deinococcus bacteria, or IS-mediatedgenetic modification of Deinococcus bacteria.

An object of the invention therefore relates to a method for introducinga nucleic acid into the genome of a Deinococcus bacterium, comprisingintroducing said nucleic acid into said genome by IS-mediated insertion.In preferred embodiments, the nucleic acid is introduced into the genomeof the bacterium by homologous recombination with an IS present in thegenome, by intron-mediated insertion into an IS, or by IS-mediatedtransposition.

A further object of the invention resides in a method for producing arecombinant Deinococcus bacterium comprising one or several copies of agene of interest inserted into its genome, the method comprisingintroducing said gene of interest into the genome of said bacterium byIS-mediated insertion and, optionally, amplifying the copy number bysubjecting said bacterium or a descendant thereof to a geneamplification treatment.

The invention also relates to a method for inducing (or increasing)chromosomal rearrangement(s) or shuffling in a Deinococcus bacterium,the method comprising expressing (or increasing expression of) at leasta transposase gene in said bacterium.

The invention also relates to a Deinococcus bacterium obtained byIS-mediated insertion of a nucleic acid, or a descendant of saidbacterium.

Another object of the invention is a Deinococcus bacterium comprisingone or several copies of a nucleic acid inserted into an IS element.

A further object of the invention resides in a nucleic acid moleculecomprising a gene of interest flanked, on one or both sides, by (i) asequence homologous to a sequence of Deinococcus IS element or (ii) asequence of an inverted repeat sequence of Deinococcus IS element.

The invention also relates to a recombinant Deinococcus bacteriumcomprising one or several copies of a transposase gene under control ofa promoter.

The invention may be performed with any Deinococcus bacteria and can beused to engineer bacteria with improved genotypes or phenotypes,particularly bacteria which express recombinant genes of interest.

LEGEND TO THE FIGURES

FIG. 1: Gene insertion by IS-mediated homologous recombination inDeinococcus.

FIG. 2: Insertion of ethanol pathway DNA construct into IS66 of D.geothermalis.

FIG. 3: Gene insertion by IS-mediated artificial transposition inDeinococcus.

DETAILED DESCRIPTION OF THE INVENTION

The invention relates to IS-mediated gene insertion or chromosomeengineering in Deinococcus bacteria, the resulting bacteria, and theuses thereof.

Insertion Sequences (IS) are transposable genetic elements identified incertain prokaryotic organisms. They have a typical length ranging from300 to 3000 bp (Mahillon and Chandler, 1998; Chandler and Mahillon,2002). In bacteria, IS are frequently found as part of natural plasmids.IS typically possess one or two open reading frames (ORFs) that encode atransposase, an enzyme that is necessary for their transposition. This(these) ORF(s) is (are) surrounded by linker regions that frequently endwith short-terminal inverted repeats (IRs) ranging typically from 7 to50 bp in length. Some IS may carry multiple repeated sequences at bothends, which may represent transposase binding sites (Zita Nagy andMichael Chandler, Research in Microbiology 155 (5) p 387-398). Contraryto transposons, IS do not contain ORF encoding drug resistance. Uponinsertion, IS often undergo short directed repeats from 2 to 14 bpimmediately outside the IRs.

Despite sequence divergence, IS have been grouped into families based onsimilarities and identities in the primary sequence of theirtransposases (Tpases) and in their genetic organization (Robinson, Lee,and Marx, 2012). This includes the disposition of their open readingframes (ORFs), the length and similarity of the terminal invertedrepeats, and the characteristic number of base pairs in the target DNAwhich they duplicate upon insertion (Mahillon, Léonard, and Chandler,1999).

Depending on the IS, insertion may be target site-selective (Craig,1997; Tobes and Pareja, 2006). Through transposition, IS can interruptthe coding region of a gene, or disrupt promoter regions and alter geneexpression. Given that there can be several copies of the same IS in agenome, IS can also serve as sites of DNA rearrangements such asdeletions, duplications and inversions of adjacent DNA segments throughhomologous recombination (Robinson et al., 2012). Insertion sequencescontribute to the variability of the prokaryotic genomes and phenotypes,and are thought to play an important role in the adaptability ofprokaryotes to the environment (Schneider and Lenski, 2004).

IS elements have been identified in D. radiodurans (Makarova et al.,2001; Islam et al., 2003; Mennecier, Servant, Coste, Bailone, andSommer, 2006; Pasternak et al., 2010). The present invention disclosesthe characterization of particular IS sequences in Deinococcus bacteria,as well as the uses thereof for genetic modification or shuffling ofsuch bacteria.

More specifically, the inventors analyzed the presence and occurrence ofdifferent IS families in Deinococcus sp. genomes. A total of 11 ISfamilies were found in 5 tested Deinococcus species, which are presentedin Table 1. IS families IS4, IS5, IS1 and IS701 were found to be themost scattered among Deinococci, the largest family being IS4, whichcontains a total of 68 members in Deinococci. Surprisingly, the genomesof D. geothermalis DSM11300 and D. geothermalis MX6-1E possess a largernumber of IS elements compared to the other Deinococcus strains (Table1). In these strains, seventy six IS elements belonging to 10 distinctfamilies were detected (DSM11300 strain) and fifty five spread into 7 ISfamilies (MX6-1E strain).

The present invention therefore shows an unexpectedly high level numberof insertion sequences in thermophile Deinococcus strains such as D.geothermalis (Table 1). This discovery brings new tools for geneticmanipulation of thermophile Deinococcus strains and, in particular,allows multicopy integration of genes into Deinococcus bacteria toincrease their expression. The presence of different IS families in asingle Deinococcus genome even allows the introduction of different DNAconstructs (one type of construct targets one family of IS). The presentinvention therefore provides a novel method to insert or spread oramplify a desired gene into a Deinococcus genome using Deinococcusinsertion sequences.

The present invention also provides a method for chromosomal engineeringof a Deinococcus bacterium by expressing or overproducing in saidbacterium a transposase that targets an IS present (preferably inseveral copies) on the chromosome or a plasmid of said bacterium.

Definitions

Within the context of the present invention, the term “InsertionSequence” or “IS” element designates a transposable genetic elementcomprising at least one transposase gene and a flanking terminalInverted Repeat. IS are devoid of drug resistance gene. IS have atypical length ranging from 300 to 3000 bp, the terminal invertedrepeats (IRs) ranging typically from 7 to 50 bp in length.

Preferred IS elements for use in the invention are Deinococcus ISelements, i.e., IS elements having the sequence of an IS present in oneor, preferably, several copies in the genome of a Deinococcus bacterium.Specific examples of Deinococcus IS elements according to the inventionare IS200/IS605; IS630; IS701; IS607; IS982; IS3; IS1; IS6; IS5; IS4; orIS66. The sequence of these IS is provided in the sequence listing.

“IS-based” methods or “IS-mediated” methods designates any method forinserting a gene in a Deinococcus bacterium which uses all or part of anIS element. Insertion is typically targeted, i.e., site-specific orsite-controlled. In particular, because IS-mediated insertion generallyfollows target selectivity of the IS element, the gene insertion is notrandom but obeys the same rule.

The term “gene” designates any nucleic acid molecule (e.g., DNAfragment) of interest, such as preferably a nucleic acid comprising anORF encoding a product (e.g., RNA or polypeptide) of interest. The genemay be natural, recombinant or synthetic. A gene may be single- ordouble-stranded, typically a DNA molecule. In a particular embodiment,the gene preferably encodes a protein, such as an enzyme. The gene mayfurther comprise one or several regulatory elements operably linked tothe ORF, such as a promoter, terminator, intron, etc.

The term “chromosomal engineering” designates any modification orrearrangement of a chromosome, such as a deletion, translocation,duplication, or inversion of one or several sequences within achromosome or episome. Chromosomal engineering may result in novelchromosomes or episomes, thus creating novel biological pathways and/orgenetic diversity in bacteria, leading to, e.g., bacteria havingimproved characteristics.

IS-Mediated Genetic Modification of a Deinococcus

As indicated the invention resides in IS-mediated genetic modificationof Deinococcus bacteria, typically to produce recombinant or geneticallyimproved bacteria. The invention is particularly advantageous sinceIS-mediated insertion is effective, can be site-selective, and allowsinsertion of multiple copies of a selected gene. IS-mediated insertionmay comprise preferably the introduction of the gene into the genome ofthe bacterium by homologous recombination with a selected IS present (orinserted or amplified) in the genome of the bacterium; or byintron-mediated insertion into an IS present (or inserted or amplified)in the genome of the bacterium; or by IS-mediated transposition. Themethod may involve the use of an insertion cassette, the construction ofartificial transposon, or the retro-homing mechanism of group II intronas described below, in combination or not with DNA-damaging treatments(UV, gamma, x-irradiation). Preferably, the IS-mediated insertion of agene in a Deinococcus bacterium includes the (targeted) introduction ofsaid gene into an IS sequence present in the genome of said bacterium.

The invention may be used to genetically modify any Deinococcuscontaining an IS element, preferably any Deinococcus strain thatcontains or that can accept at least two copies of an IS element.Examples of Deinococcus host strains that can be engineered according tothe present invention include, without limitation, D. geothermalis, D.radiodurans, D. cellulosilyticus, D. murrayi, D. guilhemensis, D.aerius, D. aerolatus, D. aerophilus, D. aetherius, D. alpinitundrae, D.altitudinis, D. apachensis, D. aquaticus, D. aquatilis, D.aquiradiocola, D. caeni, D. claudionis, D. daejeonensis, D.depolymerans, D. deserti, D. erythromyxa, D. ficus, D. frigens, D.gobiensis, D. grandis, D. hohokamensis, D. hopiensis, D. humi, D.indicus, D. maricopensis, D. marmoris, D. misasensis, D. mumbaiensis, D.navajonensis, D. papagonensis, D. peraridilitoris, D. pimensis, D.piscis, D. proteolyticus, D. radiodurans, D. radiomollis, D.radiophilus, D. radiopugnans, D. reticulitermitis, D. roseus, D.saricola, D. sonorensis, D. wulumuqiensis, D. xibeiensis, D.xinjiangensis, D. yavapaiensis, and D. yunweiensis. Preferred acceptorbacteria are thermophile Deinococcus. Most preferred bacteria areDeinococcus comprising at least 2 copies of an IS element, preferably atleast 3 copies thereof. The copies may be present in the chromosome, orinduced in said chromosome.

Accordingly, the method of the invention typically comprises thefollowing steps:

a) provision of a Deinococcus bacterium that contains at least 1,preferably at least 2 copies of a target IS element; and,b) IS-mediated insertion of a gene into said target IS element.

Preferentially, the targeted IS is an IS present in more than one copyon the genome (chromosome and/or plasmid) of the selected Deinococcusbacterium. The targeted IS is more preferably one of the IS elementsselected by the inventors which are listed in Table 1. Theidentification of several distinct IS elements, in addition, allows thepropagation and expression of different genes of interest in aDeinococcus genome. A preferred target IS is selected from IS200/IS605;IS630; IS701; IS607; IS982; IS3; IS1; IS6; IS5; IS4; or IS66.

The Deinococcus bacterium may contain said at least 2 copies naturally,or may be treated to amplify the copy number of a target IS element,prior to step b), or thereafter.

Accordingly, the invention generally comprises the following steps:

a) provision of a Deinococcus bacterium for which genetic modificationis desired;b) selection, in said Deinococcus bacterium, of at least one target ISelement present in the chromosome of said bacterium, preferably in atleast two copies;c) optionally treating the bacterium to amplify the copy number of saidselected target IS element; andd) inserting a gene into said bacterium by IS-mediated insertion intosaid selected target IS element.

In a particular embodiment, the step of treating the bacterium toamplify the copy number of the selected target IS element is conductedafter the insertion step. Also, the amplification step can be performedboth before and after the insertion step. The treatment may comprise anytreatment which allows or increases expression or activity of atransposase and/or causes a cell stress, such as a thermal shock or anirradiation of the cells, which may be selected from UV, gamma and/or Xray irradiation, either alone or in combinations, most preferably UVirradiation(s). Irradiation treatment typically comprises subjecting themicroorganisms to one or several sequential irradiations (e.g., from 1to 5), which may be of the same or different nature, preferably of thesame nature. Repeated irradiation treatments are typically carried outat intervals of between 1 and 8 hours, preferably 3 to 5 hours, and morepreferably of about 4 hours. A particularly preferred treatmentcomprises subjecting the sample to UV, X, or gamma irradiation. Such atreatment indeed allows to amplify IS copy numbers and to stimulatechromosomal engineering. Particular UV treatments are typically ofbetween 0.5 and 400 mJ/cm², more preferably of between 1 and 200 mJ/cm²,typically between 1 and 100 mJ/cm², applied for a period of time ofabout 5″ to 5′. A preferred UV treatment is 4 mJ/cm² for 30 seconds.

During the whole process, the cells may be placed in a suitable culturemedium such as, without limitation, PGY (Bacto-peptone 10 g/L, Yeastextract 5 g/L, glucose 20 g/L) or LB (Bacto-tryptone 10 g/L, Yeastextract 2.5 g/L, Sodium chloride 10 g/L). It should be understood thatother suitable culture media are known to the skilled person (Buchananet al., 1974; Difco, 1995) or may be prepared by the skilled person fromsuch known media.

For targeted insertion by homologous recombination into an IS, the geneto be inserted (or amplified) is typically assembled as a recombinationcassette, which may be cloned or not in an appropriate vector, such aspMD66. In this regard, the invention shows that linear DNA molecules canbe used directly for transformation of Deinococcus bacteria. Therecombination cassette typically comprises the gene flanked, on one orboth sides, by an HR1 region and/or an HR2 region (of about 100-1000,more preferably about 200-700, such as 300-600, typically about 500 bpeach), said HR1 and HR2 regions being homologous, respectively, to a 5′and 3′ DNA sequence of the targeted IS element. HR1 and HR2 can be anypart of the sequence of the target IS. HR1 and/or HR2 allow insertion ofthe gene of interest into the chromosome by specific homologousrecombination. The recombination cassette may comprise, in addition,e.g., a marker gene (such as a drug resistance gene). In such a case,the two genes (gene of interest and marker gene) may be placed under thecontrol of either one promoter (operon structure) or of distinctseparate promoters. Examples of marker genes include antibioticresistance genes such as genes conferring resistance to e.g., kanamycin,chloramphenicol, bleocin, oxytetracycline, hygromycin, erythromycin,puromycin, or thiamphenicol. The recombination cassette (or the vectorcarrying the same) is introduced into the selected Deinococcus strain.Introduction may be performed using techniques such as transformation,lipofection, calcium-mediated precipitation, electroporation, etc. Thepresence of the recombination cassette or vector in the cell can beverified by e.g., detection of the gene or marker gene. Afterintroduction in the host strain, integration of the recombinationcassette in the IS target site(s) occurs by homologous recombination. Inthis regard, in a preferred embodiment, insertion is induced orstimulated by thermal shock. Indeed, upon thermal shock, the vector islost and recombinant strains expressing the gene have therefore insertedthe gene into their chromosome. The results presented in the examplesshow effective insertion by IS-targeted homologous recombination. Theyfurther show that transformation of Deinococcus is effective with alinear DNA construct. They further show that IS-targeted homologousrecombination into Deinococcus can be performed with very largerecombinant cassettes. Indeed, as shown in Example C, a recombinantcassette of more than 6 kb (e.g., comprising 4 distinct genes) can besuccessfully inserted into a Deinococcus bacterium by IS-targetedhomologous recombination.

In this regard, the invention also relates to a method for introducing aDNA into a Deinococcus bacterium, the method comprising:

-   -   providing a linear DNA molecule, and    -   introducing said molecule into a Deinococcus bacterium.

More preferably, the linear DNA molecule comprises an HR1 and/or HR2region as defined above and the method further comprises a step ofmaintaining the Deinococcus under conditions allowing homologousrecombination.

In a particular embodiment, the linear DNA molecule comprises more than2 kb, even more than 3, 4, 5 or even 6 kb.

In an alternative embodiment, IS-mediated insertion is performed byconstruction of an artificial transposon containing the gene, andintroduction of the transposon into the selected Deinococcus bacterium,leading to IS-mediated insertion into the chromosome. The artificialtransposon preferably comprises the gene, a transposase gene of aDeinococcus IS element, optionally a marker gene, and one or two IRelements of a Deinococcus IS element. The artificial transposon of thepresent invention can be constructed using regions or sequences of anyIS element as described in Table 1 as starting material. Preferably, thetransposase gene and IR sequences are derived from (e.g., have asequence of a domain of) a same IS element. The transposase gene may belocated inside the artificial transposon, that is the transposase genemay be located between the two inverted repeats. Alternatively thetransposase gene may be located outside the artificial transposon. Inthat case, it may be on the transposon-carrying vector, or on thechromosome, or on a distinct vector. The expression of the transposasemay be under the control of its own promoter, a constitutive promoter,or an inducible promoter. The marker gene, when present, can be e.g.,any gene conferring resistance to an antibiotic such as kanamycin,chloramphenicol, bleocin, oxytetracycline, or hygromycin. Theconstructed artificial-transposon is typically cloned into a suitablevector, such as pmD66, and introduced into the selected Deinococcus hoststrain. Upon introduction, IS-mediated insertion of the transposonoccurs. If desirable, DNA-damaging agents such as gamma and/orX-irradiations and/or UV treatments or, more generally, any treatmentallowing or increasing expression or activity of a transposase, may beapplied to enhance the transposition and increase integration andamplification of the artificial transposon in the Deinococcus hostcells. Indeed, it has been shown that irradiation can inducetransposition in Escherichia coli (Eichenbaum and Livneh, 1998).

Another alternative embodiment for performing IS-mediated multicopyinsertion of a gene into the chromosome of a Deinococcus bacterium is touse group II introns targeting IS sequences. Mobile group II introns arecatalytic RNA elements present in a wide range of prokaryotic andeukaryotic organisms (Michel and Feral, 1995). Some of these introns canmobilize autonomously at a high frequency to allelic sites in a processknown as homing. Mobile group II introns possess an intron-encodedprotein (IEP) that has reverse transcriptase, RNA splicing (“maturase”),and DNA endonuclease activities (Frazier, Filippo, Lambowitz, and Mills,2003). Mobility initiates when the IEP helps the intron RNA fold intothe catalytically active RNA structure to promote splicing, resulting inligated exons and an intron lariat-IEP ribonucleoprotein (RNP) complex.The RNP complex recognizes specific DNA target sites and promotesintegration by reverse splicing of the intron RNA directly into onestrand of the target DNA. The IEP then cleaves the opposite strand anduses it as a primer for target DNA-primed reverse transcription of theinserted intron RNA. The resulting cDNA copy of the intron is integratedinto genomic DNA by cellular recombination or repair mechanisms. DNAtarget site recognition by the RNP complex involves the base pairing ofintron sequences denoted EBS1 and -2 (exon binding sites 1 and 2) and δto sequences denoted IBS1 and -2 (intron binding sites 1 and 2) and δ′in the DNA target site.

The EBS sequences can be mutagenized to retarget the intron to invade aselected IS sequence in Deinococcus. In the present invention, a plasmidsuch as pMD66 carrying the IEP protein (reverse transcriptase)-encodinggene and the group II intron which contains in its sequence a multiplecloning site allowing the cloning of the gene of interest was used. Theexpression of group II intron harboring the gene of interest iscontrolled by a constitutive or inducible promoter, such as the T7constitutive promoter whereas the IEP (reverse transcriptase)-encodinggene is under the control of an inducible or constitutive promoter.Alternatively, the group II introns harboring the gene of interest isintegrated into the Deinococcus chromosome and the reverse transcriptaseencoding-gene carried by a replicative plasmid is under the control ofeither a constitutive or inducible promoter. This system that usesintron-mediated insertion or amplification does not rely on homologousrecombination to achieve multicopy integration and facilitate stablechromosomal gene delivery without selection (Rawsthorne, Turner, andMills, 2006).

A further object of the invention resides in a method for producing arecombinant Deinococcus bacterium comprising one or several copies of agene of interest inserted into its genome, the method comprisingintroducing said gene of interest into the genome of said bacterium byIS-mediated insertion and, optionally, amplifying the copy number bysubjecting said bacterium or a descendant thereof to a geneamplification treatment.

The invention also relates to a Deinococcus bacterium obtained byIS-mediated insertion of a nucleic acid molecule (e.g. DNA fragment), ora descendant of said bacterium.

The invention further relates to a Deinococcus bacterium comprising oneor several copies of a nucleic acid molecule (e.g. DNA fragment)inserted into an IS element.

A further object of the invention is a nucleic acid molecule (e.g. DNAfragment) comprising a gene of interest flanked, on one or both sides,by a sequence homologous to a sequence of Deinococcus IS element, aswell as a vector comprising such a nucleic acid.

Still another object of the invention is a nucleic acid molecule (e.g.DNA fragment) comprising a gene of interest flanked, on one or bothsides, by a sequence of an inverted repeat sequence of Deinococcus ISelement, as well as a vector comprising such a nucleic acid molecule.

Chromosomal Engineering

As indicated above, another object of the invention resides in a methodfor inducing (or increasing) chromosomal engineering (e.g.,rearrangement or shuffling) in a Deinococcus bacterium, the methodcomprising expressing in said bacterium a transposase gene. Moreparticularly, the method comprises:

a) causing or inducing expression of at least one transposase in aDeinococcus bacterium; andb) selecting a Deinococcus bacterium of step a) having a reengineeredchromosome.

In step a), the transposase may be expressed on a vector, or chromosome,or supplied as a protein. The transposase is preferably a transposase ofan IS element present in said bacterium. Expression of the transposasemay be combined with a treatment of the cells to amplify a gene copynumber, such as irradiation.

For example a strain expressing one or several transposases can besubmitted to an increasing selection pressure (e.g., increasing ethanolconcentration to strengthen its resistance to ethanol). A shuffling ofthe genome will occur due to the expression of the transposases and themost resistant clones will be selected.

The method of the invention may be used to insert any gene of interestinto a Deinococcus strain, in one or more copies, allowing an increaseof its expression. The gene may encode any product of interest, such asan RNA (mRNA, tRNA, siRNA, etc.) or a polypeptide (protein, peptide,etc.). Examples of such polypeptides include, without limitation,enzymes involved in metabolism, any biologically active polypeptide,etc.

The polypeptide may be a polypeptide having pharmaceutical and/oragro-chemical interest. In a particular embodiment, the polypeptide is apharmaceutical compound (e.g., suitable for use in human or veterinarymedicine). Specific examples of such a compound include, withoutlimitation, antibiotics, bacteriostatic compounds, anti-metabolite,chemotherapeutic compounds, antioxidants, anti-inflammatory,polysaccharides, anti-parasitic agents, anti-fungal agents, anti-viralcompounds, cytokine-activity compounds, cell-growth factors, hormones,anti-depressives, anti-migraine, anti-asthma, contraceptives,anti-diabetics, psychotropic, anti-arrythmics, enzyme-inhibitors, oradjuvants.

The polypeptide may also have utility e.g., in cosmetics or agriculture,such as pigments, insecticides, pesticides, chemical-degradingcompounds, etc.

Examples of enzymes include biomass-degradation enzymes or fermentationenzymes, such as laccases, xylanases, amylases, ADH (alcoholdehydrogenase), PDC (pyruvate decarboxylase), etc. Further examples ofpolypeptides include enzymes of biological biosynthetic pathways, inparticular, enzymes involved in the synthesis of antibiotics.

Further aspects and advantages of the invention will be disclosed in thefollowing Examples section, which is illustrative.

EXAMPLES

A. Characterization of Deinococcus IS Sites

A search and compilation of IS sequences present in Deinococcus strainswas performed by the inventors. A complete list of all identified ISsequences found in different Deinococcus species is presented in Table 1below.

TABLE 1 IS distribution among Deinococcus species Total GenomeDeinococcus sp. IS200/IS605 IS630 IS701 IS607 IS982 IS3 IS1 IS6 IS5 IS4IS66 of IS Size (Mb) D. deserti 0 2 1 0 2 5 0 0 1 4 0 15 3.86 VCD115 D.geothermalis 3 1 17 1 1 0 19 8 12 8 6 76 3.25 DSM11300 D. geothermalis 60 6 0 0 0 12 2 1 18 10 55 3.25 MX6-1E D. maricopensis 0 0 0 0 0 0 0 0 10 0 1 3.5 DSM21211 D. proteolyticus 0 0 0 0 2 0 0 0 5 13 0 23 2.89 MRPD. radiodurans 9 10 0 0 0 0 0 0 2 25 1 47 3.28 Total number of 18 13 241 5 5 31 10 22 68 17 217 — IS among Deinococcus sp.

B. Gene Insertion by IS-Mediated Homologous Recombination in Deinococcus

We built Deinococcus plasmids (pMD66-type for replicative, pUC-type fornon-replicative) harboring IS-targeting cassettes (see FIG. 1). A DNAfragment containing a gene encoding resistance to hygromycin (greyrectangle) under the control of its own promoter was flanked by two 500bp regions named HR1 and HR2 (black rectangle), that are homologous toN-term and C-term sequences, respectively, of the insertion sequenceIS66 of Deinococcus geothermalis. The nucleic acid sequences of HR1 andHR2 are provided as SEQ ID NOs: 11 and 12, respectively. The IStargeting vector is then transformed into D. geothermalis and strainsexpressing the hygromycin gene are selected using the embedded marker onPGY-agar plate containing 800 μg/ml of hygromycin. In an alternativeexperiment, a linear DNA fragment containing HR1-hygromycin-HR2 was useddirectly to transform Deinococcus cells, the selection of recombinantsbeing carried out as described above. The clones that showed resistanceto hygromycin were selected. Such clones are recombinant bacteria havinginserted the target nucleic acid into an IS element. In this regard, inorder to verify the insertion of the hygromycin resistance-encoding geneinto the IS66, the clones were subjected to PCR amplification withprimers designated “A” and “B”, which anneal specifically to the regionupstream the IS66 and to the 5′-end of hygromycin gene, respectively,generating a DNA PCR fragment of about 700 bp (see FIG. 1).

The results presented in FIG. 1 confirm the insertion of the recombinantnucleic acid into the targeted IS.

C. Ethanol Pathway Genes Insertion by IS-Targeted HomologousRecombination in Deinococcus geothermalis

An IS-targeting cassette was constructed comprising a PDC gene and twoalcohol dehydrogenase encoding genes. The cassette also comprises anantibiotic resistance marker to hygromycin. The cassette is flanked bytwo 500 bp regions HR1 (SEQ ID NO: 11) and HR2 (SEQ ID NO: 12) (FIG. 2,black rectangle), that are homologous to N-term and C-term sequences ofthe insertion sequence IS66 of Deinococcus geothermalis, respectively.The cassette comprises 6548 bp. The IS-targeting cassette (linearmolecule), or a vector containing the cassette, are transformed into D.geothermalis and recombinant strains having inserted the cassette intotheir chromosome are selected using the embedded marker on PGY-agarplate containing 800 μg/ml of hygromycin. The mapping of the cassette inthe genome is confirmed by PCR using primers “C” and “D”, primer C beingspecific of each upstream sequence of IS66 and D being specific of 5′end of PDC. The results are presented in FIG. 2. They show theconstruction has been found integrated into four different loci of IS66(CDS_1696, CDS_1721, CDS_2881 and CDS_2557 (FIG. 2)), into thechromosome or into native plasmid 1 of Deinococcus.

These results confirm the efficacy of the method with linear DNAconstruct. They confirm the specificity of the method since the cassetteis found in the targeted IS. They also demonstrate the efficacy of themethod with very large expression cassettes.

D. Gene Insertion by IS-Mediated Artificial Transposition in Deinococcus

An IS-propagating cassette is prepared (FIG. 3). The gene of interest isflanked by 2 Inverted repeat sequences (black, IRs) that can berecognized by a Transposase. A Deinococcus replicative vector allowingfor Transposase thermosensitive-expression is transformed into theGOI_IRs strain. Upon Transposase expression (grey), the gene is insertedand propagated into the chromosome.

REFERENCES

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We claim:
 1. A Deinococcus bacterium comprising one or several copies ofa gene of interest inserted into an insertion sequence (IS) element. 2.The Deinococcus bacterium of claim 1, wherein said bacterium is producedby a method comprising introducing in a targeted manner said gene ofinterest into an IS present in the genome of said bacterium and,optionally, amplifying the copy number by subjecting said bacterium or adescendant thereof to a gene amplification treatment.
 3. The Deinococcusbacterium of claim 1, wherein the insertion sequence is present inseveral copies in the genome of said bacterium.
 4. The Deinococcusbacterium of claim 1, wherein the IS is selected from IS200/IS605;IS630; IS701; IS607; IS982; IS3; IS1; IS6; IS5; IS4; or IS66.
 5. TheDeinococcus bacterium of claim 1, wherein the gene of interest isflanked, on one or both sides, by an insertion-mediating sequence. 6.The Deinococcus bacterium of claim 5, wherein the insertion-mediatingsequence comprises a sequence homologous to a sequence of the IS,allowing insertion of the gene of interest into the IS element byhomologous recombination.
 7. The Deinococcus bacterium of claim 5,wherein the insertion-mediating sequence comprises an Inverted Repeatelement of an IS, allowing insertion of the gene of interest into the ISelement by transposition or an intron sequence, allowing insertion ofthe gene of interest into the IS element by retro-homing.
 8. TheDeinococcus bacterium of claim 1, wherein the gene of interest comprisesan open reading frame encoding a biologically active polypeptide or RNA.9. The Deinococcus bacterium of claim 1, wherein the gene of interestcomprises an open reading frame encoding a polypeptide havingpharmaceutical and/or agro-chemical interest or which is an enzyme. 10.The Deinococcus bacterium of claim 9, wherein the polypeptide is apharmaceutical compound.
 11. The Deinococcus bacterium of claim 1,wherein the Deinococcus bacterium is selected from D. deserti; D.geothermalis; D. maricopensis; D. proteolyticus; D. radiodurans; D.murrayi; D. cellulolysiticus; D. guilhemensis; D. aquaticus; D. ficus;D. gobiensis; D. grandis; D. radiopugnans; or D. roseus, or anythermophile Deinococcus bacterium.
 12. A method of producing apharmaceutical and/or agro-chemical polypeptide of interest or an enzymecomprising culturing a bacterium according to claim 9 under conditionsthat permit the expression of a polypeptide encoded by a gene ofinterest encoding said pharmaceutical and/or agro-chemical polypeptideor said enzyme.